The present invention relates to methods and devices for determining analytes in a liquid.
The analytical detection and determination of the concentration of certain biologically and medically relevant substances from complex samples is a basis for modern medical diagnostics. In recent years methods and processes have been developed to obtain exact analytical results with sample volumes that are becoming smaller and smaller especially by the introduction of microanalytical methods. The “lab-on-a-chip” systems that are being used to an increasing extent operate with quantities of liquids in the micro to nano liter range which have to be moved in these systems to a spatially defined analytical area which is the site of the examination. The actual determination of the analyte is then carried out at these sites, usually with the aid of specific sensors.
Conventional “lab-on-a-chip” systems generally consist of microstructured closed channels which transport the liquid to the actual sensory elements. Mechanical micropumps or electrokinetic methods are usually used to move the liquids. Thus liquids can for example be moved in these channels by electroosmosis, hydrostatic pressure differences, capillary forces or centrifugal forces. Other methods for transporting very small amounts of liquid are electrowetting as described for example in “Electrostatic Actuation of Liquid Droplets for Microreactor Applications” (Washizu M. in IEEE Transactions of Industry Applications 34(4):732-737, 1998), “Creating, Transporting, Cutting and Merging Liquid Droplets by Electrowetting-Based Actuation for Digital Microfluidic Circuits” (Cho S. K., Moon H. Kim C. J. in Journal of Microelectromechanical Systems 12(1):70-80, 2003) or “Micropumping by electrowetting” (Kim C. J. in Proceedings of 2001 ASME International Mechanical Engineering Congress and Exposition, Nov. 11-16, 2001, New York, N.Y.), and the transport of liquids on surfaces with the aid of acoustic surface waves, so-called surface acoustic waves (SAW) as described for example in “Flatland fluidics” (Wixforth A., Scriba J. and Gauer C. in mstnews 5/02, pages 42-43).
Analytes are usually determined in microanalytical systems with the aid of sensors that are integrated in the channels of the chips. The measuring methods of these sensors in the previously used microanalytical methods are based in particular on spectroscopic methods such as fluorescence or absorption measurements, electrochemical methods, conductivity, luminescence or electrochemiluminescence methods and detection methods using waveguide sensors. In contrast, biosensors, ion-selective electrodes and other sensors that are widely used in macroscopic routine diagnostics have hitherto proven to be unsuitable for routine use in microanalytical systems. The reasons for this are, in particular, the high manufacturing costs of such microstructured sensors and electrodes and the fact that so far no satisfactory method has been found to move liquids in these systems by active pumping from outside. Other microanalytical devices are in particular protein arrays and arrays for determining nucleic acids. Furthermore, there are sensor modules which are incorporated into clinical and/or chemical analyzers. These are especially modules for determining electrolytes and other analytes such as glucose or lactate. However, these methods that are established in laboratories usually use considerably larger sample volumes.
The microanalytical devices that are commonly used are almost exclusively composed of microfluidic channels with the exception of arrays for protein and nucleic acid analysis. These closed channels have a width and depth of a few micrometers but are usually very long so that the volumes of these channels is large relative to their cross-section. Consequently, a considerable proportion of the sample volume in these systems cannot be used to determine the analyte in the sensory areas of the system and represents an unusable dead volume. Thus there are fundamental limits to a further reduction of the required amount of sample in these channel systems. Furthermore, such channels have the major disadvantage that the surface which is in direct contact with the sample is very large relative to the volume. Thus there is a high probability that components of the liquid will remain behind on the surface of the channels and can thus contaminate samples which are moved in the same channels for subsequent measurements. Hence such systems can often only be used as disposable articles due to the said carry-over problems. Another disadvantage of such microanalytical systems is that mixing liquids in microchannels is either impossible or very complicated and air bubbles that may occur can easily bring the flow in the channels to a standstill. Hence such systems are relatively trouble-prone and expensive to manufacture so that for cost reasons they often have to be used several times in routine operations which, however, for the above-mentioned reasons (carry-over problems) is at present not possible.
At present, ion-selective electrodes are used above all in macroscopic analytical systems and especially in modules for electrolyte analysis in clinical and chemical analytical systems. Such macroscopic detection systems have considerable disadvantages. Thus in addition to the considerably larger sample volumes, such modules and systems require numerous tubes, valves and pumps to control the flow of liquids within these systems. For example, air segments have to be introduced into the stream of liquid in order to clean the tubes and sensors between individual measurements and calibrations. Additional sensors and, in particular, light barriers or conductivity sensors are required to control the liquid flows in order to ensure that the air segments are correctly introduced and discharged. Although, like the microanalytical systems with microfluidic channels, only a relatively small volume is necessary for the actual determination of the analyte, an approximately 20-fold larger volume of liquid has to be used in the current systems in order to ensure a measurement that is free from carry-over. Hence such systems are often very susceptible to faults and require a large amount of maintenance. The construction described above does not allow the manufacture of instruments that are easy to handle and portable which could be used ideally for a doctor's laboratory or near patient diagnostics. Another disadvantage of the instruments described above is their high manufacturing costs since all systems and modules have to be assembled from many different components. In contrast to macroscopic analytical systems, there are at present no ion-selective electrodes for microanalytical methods and devices which are suitable for multiple measurements in routine operation like their macroscopic analogues.
Microarrays are a special case of microanalytical systems. Microarrays are understood as analytical systems which have many sensory elements on a support substance that are usually arranged at regular distances to one another so that they can be used for many simultaneous or staggered determinations. Microarrays are used especially to analyse proteins and nucleic acids. It is difficult to regenerate such arrays and hence such systems are also not suitable for multiple use for the above-mentioned reasons.
Some microarrays for protein determination operate with planar surfaces. However, these arrays require relatively large volumes. Thus, for example, about 50 μl sample liquid has to be incubated in such systems in order to allow the analyte to bind to the detection molecules. In order to prevent a depletion of the analyte, the sample has to be mixed thoroughly which is a major technical problem.
All these arrays are intended to be used only once. Usually, flat arrays with large volumes are used in which mixing during incubation is also a technical challenge. The analyte is usually detected by optical methods which require expensive and complex optical detection systems so that these detection methods can only be carried out in a few special laboratories with high quality technical equipment.
Methods and devices have been described to solve these technical problems which can transport liquids especially in microanalytical systems.
The German laid-open document DE 10117771 A1 describes methods and devices for manipulating small amounts of liquids with the aid of acoustic surface waves. The object of this patent application described in the laid-open document is to localize and optionally to mix liquids on a solid chip. For this purpose devices and methods are described which can move liquids by means of acoustic surface waves over a flat substrate towards so-called functionalized areas. A chemical or biological reaction can, for example, take place in such functionalized areas. For this purpose, DE 10117771 A1 describes devices in which such functionalized areas are located at certain sites directly in or on the surface of the solid chip which, among others, can be used as sensors in analytical methods. The functionalized areas for analysing the liquid are directly integrated into the substrate of the solid chip on which the liquid is transported, i.e., the devices that are relevant for liquid transport and the devices that are relevant for determining the analyte in the liquid are combined in a single plane, the transport plane.
However, it is very costly and technically complicated to manufacture and also to purify such multifunctional surfaces and hence such systems can neither be used as disposable articles nor in routine analytics. Furthermore, the sensors integrated into the surface of the carrier chips represent inhomogeneities in the surface of the carrier substrate, for example, due to different surface wetting properties or spatial elevations or depressions. This greatly limits the uniform transport of liquids over the surface of the carrier substrate and thus complex controls and/or additional forces are required to compensate for these inhomogeneities and to enable a uniform and effective transport of the liquid.
DE 10117771 A1 also describes arrangements in which two solid surfaces oppose one another and between which the liquid to be examined is located and in contact with both surfaces. In this case, the devices for generating the acoustic surface waves and the functionalized areas can be present on the two different surfaces. However, even in such arrangements the transport of liquid on the substrate of the transport plane is not independent of the functionalized areas since the liquid volume is always in contact with both surfaces. Additional interactions occur with such arrangements and, in particular, surface effects, interfacial effects and capillary effects between the liquid and the two contacted surfaces and, hence, such arrangements are usually not suitable for transporting liquids over the substrate but can be used especially to mix a liquid.